Vibrio cholerae is a facultative pathogen and the causative agent of cholera. Hallmarks of the disease include profuse watery diarrhea resulting from the action of secreted cholera toxin, and deadly, explosive outbreaks. The strong link between epidemics and human overcrowding in areas with untreated drinking water suggests an efficient mode of fecal-oral transmission via an aquatic intermediate. In support of this notion, we discovered that V. cholerae exit cholera victims in a heightened state of transmissibility (referred to as hyperinfectivity), which persists for several hours after shedding into pond water. Knowledge of the molecular basis for this phenotype, and a general characterization of this transmissible form of V. cholerae, would contribute to the design of vaccines to prevent cholera at the initial stage of infection. In prior work, we discovered that motility in the absence of chemotaxis is one determinant of hyperinfectivity. To identify additional determinants aiding in transmission, we measured global transcriptional changes in human-shed V. cholerae during the transition to an aquatic microcosm. This analysis revealed a number of regulators and effectors that potentially contribute to cholera transmission. In related work, we used a genetic selection to identify genes that are `pre-induced' at late stages of infection and which subsequently play roles in the transition of V. cholerae to aquatic environments. In Aims 1 and 2 of this project, we propose to determine if genes regulated late in infection or in cholera stool play important roles in the transition to pond water and in hyperinfectivity. Transcriptional regulators with important roles in these phenotypes will be further analyzed by transcriptional profiling to define their corresponding regulons. In Aim 2, we also propose to measure and correlate changes in the transcriptome and proteome of cholera stool V. cholerae during the transition to pond water. In Aim 3, we propose to apply knowledge of the effectors of transmission to develop and test a mucosal vaccine in a mouse maternal model of immunization and challenge of pups. We hypothesize that an acellular vaccine expressing a repertoire of antigens that are stably expressed on the surface of transmissible forms of V. cholerae will provide enhanced protection to challenge by the transmissible form of this pathogen. These studies will establish a basis for understanding both the hyperinfective phenotype of V. cholerae and its transition to aquatic environments. This knowledge will enhance our understanding of transmission of this and perhaps other water-borne pathogens, helping pave the way to development of novel vaccines that target transmissible forms of facultative pathogens.